A Functional Polymorphism of the Vasoactive Intestinal Peptide Receptor 1 Gene Correlates with the Presence of HLA-B *2705 in Sardinia

ORIGINAL ARTICLE A functional polymorphism of the vasoactive intestinal peptide receptor 1 gene correlates with the presence of HLA-B *2705 in Sardinia

F Paladini1, E Cocco1, A Cauli2, I Cascino3, A Vacca2, F Belfiore3, MT Fiorillo1, A Mathieu2 and R Sorrentino1,4 1Department of Cell Biology and Development, ‘Sapienza’ University of Rome, Roma, Italy; 2Department of Medical Sciences, University of Cagliari, Cagliari, Italy; 3Cell Biology Institute, CNR, Monterotondo Scalo, Roma, Italy and 4Istituto Pasteur-Fondazione Cenci Bolognetti, ‘Sapienza’ University of Rome, Roma, Italy

The association of HLA-B27 with ankylosing spondylitis (AS) is the strongest among all inflammatory diseases. However, the exact role of these molecules in disease pathogenesis is still unknown. The existence of HLA-B27 variants rarely found in patients introduces a further level of complexity. It is now accepted that other genes of minor impact contribute to modify disease susceptibility and these genes might be diverse in different populations depending on the genetic background. We report here a study performed in Sardinia, an outlier population in which two major HLA-B27 subtypes are present, B *2705 strongly associated with AS and B *2709 which is not, and show the co-occurrence of the B *2705 allele with a single nucleotide polymorphism (SNP) mapping at 3 0-UTR of the receptor 1 (VIPR1) for the vasoactive intestinal peptide (VIP), a neuropeptide with anti-inflammatory properties. This same SNP is associated with a different kinetics of down-modulation of the VIPR1 mRNA in monocytes after exposure to lipopolysaccharide (P ¼ 0.004). This particular setting, HLA-B *2705 and a functional polymorphism in VIPR1 gene, might be due to a founder effect or might be the result of a selective pressure. Irrespectively, the consequent downregulation of this receptor in the presence of a ‘danger’ signal might influence susceptibility to AS. Genes and Immunity (2008) 9, 659–667; doi:10.1038/gene.2008.60; published online 31 July 2008

Keywords: HLA-B27; ankylosing spondylitis; VIP-receptor; Sardinia; polymorphism; 3 0-UTR

Introduction The human type 1 receptor for VIP (VIPR1) gene maps on human chromosome 3 and is highly conserved Vasoactive intestinal peptide (VIP) is a 28mer pleiotropic through species.2 Interestingly, VIPR1 has been reported neuropeptide involved in the maintenance of neuroen- to be down-modulated in cells of the immune system docrine immune systems communication.1 VIP exerts a after activation.11 This is compatible with a role for this broad range of biological functions through specific molecule in hampering the correct order of events receptors also present on immune cells. These receptors orchestrating the inflammatory response. belong to the group II of G-protein-coupled receptors, Ankylosing spondylitis (AS) is a chronic inflammatory the secretin receptor family.2 VIP, when released in the rheumatic disease exhibiting a strong genetic compo- lymphoid organs by nerve stimuli and by activated nent.12 The major susceptibility factor is HLA-B27, whose immune cells, modulates the function of inflammatory role in pathogenesis has not been fully understood.13,14 cells through its receptors, thus affecting both innate and Moreover, although HLA-B27 is present in the high adaptive immunity.3,4 Accordingly, VIP has been de- majority of patients with AS, it has been reported that the scribed as an effective therapeutic agent in several animal HLA region accounts for only 20–50% of the genetic models of inflammatory/autoimmune diseases, such as contribution calculated in more than 90% by twin inflammatory bowel disease, collagen-induced arthritis, variance model.15 Accordingly, the effect of additional experimental autoimmune encephalomyelitis, endotoxic genes of minor impact within and outside the HLA shock, Parkinson’s disease and Sjogren’s disease.5–10 region is generally acknowledged.16 However, its translation to the clinic and its application Sardinian population is considered an outlier popula- in the corresponding human diseases is still far off. tion with genetic features making it highly informative for association studies.17,18 Accordingly, AS in Sardinia co-segregates with a highly conserved HLA haplotype Correspondence: Dr R Sorrentino, Department of Cell Biology and (A2, B *2705, DR16) shared by 92% of patients.19 A Development, ‘Sapienza’ University of Rome, via dei Sardi 70, further reason that makes Sardinian population particu- Roma 00185, Italy. E-mail: [email protected] larly amenable to study AS, is the presence of two major Received 4 April 2008; revised 2 July 2008; accepted 2 July 2008; HLA-B27 alleles, B *2705 and B *2709. The latter, that published online 31 July 2008 segregates with a different haplotype, has been rarely Vasoactive intestinal peptide receptor 1 and HLA-B27 F Paladini et al 660 found in patients.20,21 Interestingly, the two HLA-B27 the same PCR fragment: rs896 and rs9677. In this case as molecules, B *2705 and B *2709, bind differently a peptide well, genotype distribution was not significantly differ- derived from VIPR1 (pVIPR1 aa 400–408)22 that could ent from that in the random controls. explain the pVIPR1-specific autoreactivity present in As a measure of whether the differences reported here B *2705 but not in B *2709-positive individuals23 (MT were not due to population stratification, genotype Fiorillo, unpublished data). Therefore VIPR1 represents distribution of an SNP mapping at 50 end (position an interesting candidate gene for association studies in –13910; rs4988235) of the gene for lactase was also AS although, in a genome-wide map, no linkage has analysed. This polymorphism has been reported to vary been found between AS and p22 region of chromosome 3 considerably from northwestern to southeastern Europe where VIPR1 gene maps.2 Accordingly, we have ana- and differences in allele frequencies between cases and lysed VIPR1 single nucleotide polymorphisms (SNPs) controls indicate population stratification.24 The results frequencies in HLA-B *2705 positive both patients with (Table 3) do not show a significant difference in the allelic AS and controls versus random controls in Sardinia. The frequencies of this SNP between random controls and the results indicate that HLA-B *2705, independently from HLA-B27 cohorts and the frequency of the C allele is the disease, co-occurs more frequently with allele T at the around 0.9 as already reported for the Sardinian SNP rs896 in the 30 UTR of VIPR1 gene (72% of HLA- population.25 B *2705 individuals possess at least one T at rs896 vs 58% of random controls, P ¼ 0.001). These data prompted us Down-modulation of VIPR1 mRNA correlates with a 30-UTR to verify whether this polymorphism correlates with a polymorphism function. The results show that the presence of allele T at VIPR1 is known to be down-modulated in immune cells rs896 strongly influences the kinetics of downregulation such as T lymphocytes or monocytes after stimulation. of the VIP receptor 1 in monocytes exposed to lipopo- Monocytes have a relevant role in inflammation and lysaccharide (LPS). respond quickly to bacterial exposure. Moreover they express VIPR1 as the only receptor for VIP.11 LPS, an endotoxin expressed by Gram-negative bacteria, is a Results powerful activator of the inflammatory response in monocytes. Monocytes from five donors were then Co-occurrence of HLA-B *2705 with allele T at SNP rs896 of exposed to LPS for 0, 3, 6, 9 and 12 h. Afterwards, the VIPR1 gene amount of VIPR1-specific mRNA has been evaluated by Allele frequencies of four SNPs mapping in the VIPR1 quantitative PCR. A high variability of VIPR1 mRNA in gene were analysed in a Sardinian control population untreated monocytes from different subjects has been (N ¼ 261). Genotype distribution was in Hardy–Wein- noticed, probably reflecting the basal activation of berg equilibrium for all SNPs. Linkage disequilibrium monocytes (Figure 1). However, two kinetics by which (LD) values of adjacent SNPs were the highest for rs896- VIPR1 mRNA was downregulated after LPS stimulation rs342511: (r2 ¼ 0.64), lower for rs437876-rs896 (r2 ¼ 0.17) could be envisaged: one in which a reasonable high level and the lowest for rs896-rs9677 (r2 ¼ 0.09). Allele and (450%) of mRNA was maintained through time (sub- genotype frequencies at the four SNPs in HLA-B *2705- jects A and C) and a second one in which the decrease of positive patients with AS (N ¼ 110) and in B *2705 the mRNA was pronounced (B, D and E). After 12 h, in controls (N ¼ 104) were similar with the only exception some cases there is a trend to restore the amount of of genotype T/T at rs9677 that is 19% in the B *2705 mRNA to the level before LPS treatment (A, D), in some controls and 30% in the patients with AS (P ¼ NS). We others remains low. The experiments were repeated therefore considered the two B *2705-positive cohorts as using monocytes from 53 donors that had been typed for a single one in the statistical analysis. Allele distribution the four SNPs along the VIPR1 gene. Nine hours time at rs896 is different in the B *2705-positive cohort point was chosen as the most representative to evaluate compared to random controls (allele C at rs896: 55 the percentage of down-modulation of the VIPR1 mRNA versus 65%; P ¼ 0.001, pc ¼ 0.01, OR ¼ 0.65; 95% CI ¼ 0.5– after LPS exposure. Depending on the number of 0.85) (Table 1). Genotype distribution at rs896 is also monocytes rescued, in some cases (n ¼ 27) the evaluation different with a significant Cochrane–Armitage test for has been performed also at 3 h time point. The trend (P ¼ 0.003). The number of subjects possessing at percentage of the specific mRNA in LPS-treated mono- least one T at rs896 is also significantly higher in the cytes was compared to untreated cells. The correlation B *2705-positive cohort than in the random controls (72 between VIPR1 mRNA and three SNPs: rs437876, rs896 versus 58%; P ¼ 0.001, OR ¼ 1.9; 95% CI ¼ 1.3–2.8). When or rs9677 genotypes has been analysed at 3 h (Figure 2a) the three SNPs (rs437876, rs896 and rs342511) showing and at 9 h (Figure 2b) after LPS stimulation. The most higher LD are taken into account, haplotype analysis significant correlation (P ¼ 0.004) takes place between the (Table 2) shows a prevalence of the GTG combination in presence of a T at rs896 and a sharp decrease of VIPR1 the HLA-B *2705-positive cohort (P ¼ 0.005) and a paral- transcript after 9 h from LPS exposure (Figure 2b). lel decrease of the GCA haplotype (P ¼ 0.01), pointing Rs9677 maps only 526 bp downstream rs896 but shows again at rs896 as the relevant polymorphism among a low LD with it. The two SNPs have been amplified in those analysed. To verify that the association was due to the same PCR fragment and processed together thus the presence of HLA-B *2705 allele, we analysed: (1) a representing a valuable reciprocal quality control: rs9677 small cohort of 58 HLA-B27-positive subjects typed as does not show any trend of correlation. The same HLA-B27 but not B *2705. As shown in Table 1, genotype analysis at 3 h after LPS exposure reveals also a distribution was very similar to that of the random correlation between the down-modulation of VIPR1 controls. (2) An additional cohort of 118 controls for and rs896 that, interestingly, is higher for the T/T than genotype distribution of the two SNPs co-amplified in for the C/T genotype, suggesting a small gene dosage

Genes and Immunity Table 1 Allele frequencies and genotype distribution of 4 SNP in random controls, HLA-B *2705 positive patients with AS and controls

SNP Random HLA-B *2705 P HLA-B *2705 P HLA-B *2705 P OR (95% CI) HLA-B27-positive Random controls controls AS patients controls individuals individuals other than (N ¼ 118) (N ¼ 261) (N ¼ 110) (N ¼ 104) (N ¼ 214) B *2705 (B *2702, B *2707, %(n) %(n) %(n) %(n) %(n) B *2709, B *2713) (N ¼ 58) %(n)

Rs 437876 A 39 (204) 40 (89) 40 (84) 40 (173) 46 (53) G 61 (318) 60 (131) NS 60 (124) NS 60 (255) NS 54 (63) AA 14 (37) 13 (14) 18 (19) 15 (33) 22 (13) GA 50 (130) 55 (61) 44 (46) 50 (107) 47 (27) GG 36 (94) 32 (35) 38 (39) 35 (74) 31 (18)

Rs 896 C 65 (338) 55 (121) 0.01 54 (113) 0.01 55 (234) 0.001 0.65 65 (75) 64 (150) T 35 (184) 45 (99) 46 (95) 45 (194) (pc ¼ 0.01) (0.5–0.85) 35 (41) 36 (86) CC 42 (109) 26 (29) 29 (30) 28 (59) Ref 40 (23) 42 (49) CT 46 (120) 57 (63) 51 (53) 54 (116) 0.006 1.8 (1.2–2.7) 50 (29) 44 (52) TT 12 (32) 16 (18) 20 (21) 18 (39) 0.006 2.2 (1.3–4.0) 10 (6) 14 (17) aocieitsia etd eetr1adHLA-B27 and Paladini 1 F receptor peptide intestinal Vasoactive Presence of T 58 (152) 74 (81) 0.01 71 (74) 0.02 72 (155) 0.001 1.9 (1.3–2.8) 60 (35) 58 (69)

Cochran–Armitage trend test 0.003 Rs 342511 al et A 60 (315) 54 (119) NS 51 (107) 0.03 53 (226) 0.02 65 (75) G 40 (207) 46 (101) 49 (101) 47 (202) (pc ¼ NS) 35 (41) AA 35 (92) 28 (31) 25 (26) 27 (57) 41 (24) AG 50 (131) 52 (57) 53 (55) 52 (112) 47 (27) GG 15 (38) 20 (22) 22 (23) 21 (45) 12 (7)

Rs 9677 C 49 (254) 47 (103) NS 54 (113) NS 50 (216) NS 46 (53) 51 (120) T 51 (268) 53 (117) 46 (95) 50 (212) 54 (57) 49 (116) CC 22 (58) 24 (26) 28 (29) 26 (55) 21 (12) 28 (33) CT 53 (138) 46 (51) 53 (55) 49 (106) 53 (31) 46 (54) TT 25 (65) 30 (33) 19 (20) 25 (53) 26 (15) 26 (31)

Abbreviations: NS, not significant; SNP, single nucleotide polymorphism. ee n Immunity and Genes 661 Vasoactive intestinal peptide receptor 1 and HLA-B27 F Paladini et al 662 Table 2 Haplotype distribution in random and HLA-B *2705-positive cohorts

Haplotypes Random controls HLA-B *2705 P OR (95% CI) rs 437876, rs896, rs342511 2n ¼ 522 2n ¼ 428

GTG 0.303 0.389 0.005 1.5 (1.1–1.9) ACA 0.341 0.321 NS GCA 0.252 0.184 0.01 0.66 (0.48–0.90) ATG 0.045 0.045 NS GCG 0.042 0.038 NS

Abbreviation: NS, not significant.

Table 3 Allele frequencies and genotype distribution of SNP rs4988235 at position –13910 of the gene for lactase in random controls and HLA-B27-positive cohorts

SNP Random controls HLA-B *2705 HLA-B *2705 controls HLA-B27-positive individuals other (N ¼ 261) AS patients (N ¼ 110) (N ¼ 104) than B *2705 (B *2702, B *2707, %(n) %(n) %(n) B *2709, B *2713) (N ¼ 58) %(n)

Rs 4988235 A 94 (492) 95 (210) 94 (195) 93 (108) G 6 (30) 5 (10) 6 (13) 7 (8) AA 89 (233) 91 (100) 87 (91) 86 (50) GA 10 (26) 9 (10) 13 (13) 14 (8) GG 1 (2) 0 (0) 0 (0) 0 (0)

Abbreviation: SNP, single nucleotide polymorphism.

Figure 1 Time–course of the down-modulation of the vasoactive intestinal peptide 1 (VIPR1) mRNA in monocytes after lipopolysaccharide (LPS) stimulation. Relative real-time quantification of mRNA after 0, 3, 6, 9 and 12 h of exposure to LPS in monocytes derived from five individuals. AU, arbitrary units.

effect on the kinetics of the mRNA downregulation. After rs896, whereas monocytes C/C homozygous at rs896 9 h however, the difference between the heterozygous maintain a high level of expression. (C/T) and the homozygous (T/T) status is not anymore detectable and the presence of T at rs896 strongly correlates with a low residual amount of VIPR1 mRNA. Discussion Common genetic variations contribute appreciably to VIPR1 protein analysis differences in phenotypes, and therefore are likely to be To verify whether the mRNA downregulation deter- involved in many complex genetic diseases. We show mines an effective lower amount of VIPR1 receptor here that one such variant (rs896) mapping at the 30-UTR protein, western blot analysis was performed using of the VIPR1 gene correlates with a different kinetics of monocytes from three subjects homozygous for C and 3 its mRNA downregulation in activated monocytes. These for T at rs896 exposed to LPS for 6, 9 and 12 h (Figure 3). mononuclear cells express a relatively high, albeit After 9 h of LPS treatment, the VIPR1 protein is variable, amount of VIPR1 mRNA as unique receptor decreased of about 40% in monocytes typed as T/T at for VIP11 and are equipped to respond promptly to

Genes and Immunity Vasoactive intestinal peptide receptor 1 and HLA-B27 F Paladini et al 663

Figure 2 Variation of vasoactive intestinal peptide 1 (VIPR1) mRNA in monocytes after lipopolysaccharide (LPS) exposure. Monocytes from random donors previously typed for rs437876, rs896 and 9677 have been harvested at time 0, 3 h (n ¼ 27) and 9 h (n ¼ 53) after exposure to LPS. The amount of VIPR1 mRNA was determined by relative quantitative PCR. (a) The percentage of mRNA variation between time 0 and 3 h and (b) between 0 and 9 h in relation to genotype distribution of rs437876, rs896 and rs9677 is reported. Median values and interquartile ranges are indicated. bacterial, inflammatory stimuli. When stimulated with demonstrated in T lymphocytes after a strong stimulus LPS, they are able to down-modulate the expression of such as the engagement of CD3 with a specific anti- VIPR1 transcript in a manner that correlates with the body.11 This has been correlated with the anti-inflamma- rs896 variation. VIPR1 down-modulation has been also tory properties of VIP as signalling through VIPR1

Genes and Immunity Vasoactive intestinal peptide receptor 1 and HLA-B27 F Paladini et al 664 mechanism, as small changes in mRNA half-life result in a remarkable variation in the mRNA available for translation into functional protein as also shown here.35–38 Notably, the same allele associated with a more efficient downregulation is also significantly increased in the cohort of HLA-B *2705-positive individuals in Sardinia, an allele strongly associated with AS. How HLA-B27 influences disease pathogenesis is unknown, but it is becoming more and more evident that several distinct features of these molecules can be involved. Our findings of the co-occurrence of HLA-B *2705 allele with a more efficient downregulation of the VIPR1 mRNA and, therefore, with a lower amount of receptor available for VIP and its anti-inflammatory properties, could influence disease evolution. It must be noticed however, that this association is not limited to patients, but stands also for the B *2705-positive cohort with no sign of disease. It is also of note that in a small cohort of 58 individuals carrying B27 alleles other than B *2705 (mainly B *2709), we did not find any difference from the controls in the genotype distribution of rs896, reinforcing the idea that Figure 3 Western Blot analysis of vasoactive intestinal peptide 1 the association of allele T at rs896 is HLA-B *2705- (VIPR1). (a) Expression of VIPR1 protein in monocytes from subjects specific. This particular setting of an HLA haplotype genotyped as T/T or C/C at rs896 after 0, 6, 9 and 12 h after and a functional polymorphism in a gene involved in the lipopolysaccharide (LPS) exposure as detected by western blot regulation of the immune response but mapping in a analysis. One representative experiment out of three is shown. (b) VIPR1 protein levels were evaluated by densitometric analysis different chromosome could be due to a founder effect or as described in ‘Materials and methods’. Values are the mean±s.d. can be the result of a selection operated in Sardinia by of three independent experiments and are reported as percentage of endemic parasite diseases such as malaria.39 This would 100% VIPR1 expression arbitrarily attributed to the untreated cells. not be surprising as it has been demonstrated that several genes have undergone to selection by Plasmodium falciparum endemic in Sardinia, among which immune inhibits nuclear factor-kB activation.26 Accordingly, VIP response genes and that this might represent a favour- has been reported to protect mice from lethal endo- able immunogenetic background for some autoimmune toxemia, presumably by downregulating endogenous diseases, as suggested for multiple sclerosis.40 Note- pro-inflammatory macrophage-derived mediators,27 to worthy, a recent paper has reported a genetic association inhibit the production of several pro-inflammatory between polymorphisms mapping at the 30 end of the cytokines28–31 and chemokines such as IL-8,26 and to VIPR1 gene and rheumatoid arthritis34 confirming both exert a protective effect in some models of autoimmune the relevance of this region in the regulation of the VIPR1 diseases.5–10 It has been speculated that the suppressive gene expression and of the VIP/VIPR1 signalling path- effect on VIPR1 transcription can be mediated by a way in modulating the tolerance in rheumatic, inflam- cis-regulatory repressor element located on the gene. matory diseases. Pei cloned a repressor protein that downes VIPR1 expression in rats,32 however, a homologous VIPR1 repressor protein in humans has not been identified as Materials and methods yet. It has been postulated that the down-modulation of VIPR1 after T-cell activation may be mediated by Ikaros Subjects transcription factor.33 Our data show that the 30-UTR also A total of 110 HLA-B *2705-positive patients with has an important role. This is not surprising, since post- diagnosis of AS according to the modified New York transcriptional regulation provides a mean to rapidly criteria have been selected in Cagliari, Sardinia. The alter gene expression in response to diverse stimuli, and majority of them has been reported in other studies.20,21 it is frequently observed in genes encoding proteins with Random controls (N ¼ 261) and HLA-B *2705 positive essential cellular functions such as proliferation and (N ¼ 104) were blood donors recruited from Sardinia. stress response. It is possible that rs896 is a marker for Patients with AS were 75% men and controls were this association as there are other SNPs in the region therefore balanced for gender (72% men). All patients showing a complete or a very strong LD with it.34 and controls gave their informed consent. Two further Noteworthy however, SNP rs896 (C/T) is located in the cohorts of 118 controls and 58 HLA-B27 positive other middle of a stretch of UA-rich sequence (UUUUU/ than B *2705 individuals were subsequently selected. The CAAA) where a C interrupts the UA sequence. This latter were as follows: 37 were B *2709, 11 were B *2702, 8 interruption is likely to modify the RNA secondary B *2707 and 2 B *2713. Of these, the majority (N ¼ 46) structure and possibly the binding of a protein that were healthy individuals and only 12 patients with AS, might be induced by LPS, as the effect of this as the high majority of the latter are typed as B *2705. polymorphism, already visible after 3 h, becomes more evident at 9 h after LPS exposure. This timing is in SNP typing agreement with what reported for T lymphocytes.11 The The following SNPs: rs437876 (A/G) in intron 4; rs896 control of mRNA stability is a potent regulatory (C/T), rs342511 (A/G) and rs9677 (C/T) in the 30-end

Genes and Immunity Vasoactive intestinal peptide receptor 1 and HLA-B27 F Paladini et al 665 region of the gene were analysed. Amplification of 37 1C in a humidified 7% CO2 incubator in the presence human genomic DNA was performed in a PCR reaction of LPS (0.05 mgml1) for the indicated time. mixture (30 ml) containing: 1.2 mM of primers, 100 ng of genomic DNA, 1 mM of MgCl2, 0.2 mM deoxyribonucleo- Quantitative PCR analysis tide triphosphates (dNTPs) and BioTaq polymerase (1 U; Total RNA was isolated from cell pellets by TRIzol Bioline, London, UK). PCR amplifications were allowed reagent (Invitrogen, Carlsbad, CA, USA) as recom- to proceed with an initial melt (95 1C, 5 min), followed by mended. After denaturation of freshly prepared RNAs 35 cycles of melting (95 1C, 30 s), annealing (40 s) and (approximately 2 mg) at 65 1C for 5 min, SuperScript III extension (72 1C, 40 s), and final extension at 72 1C Reverse Transcriptase (Invitrogen) was used for oligo- (7 min). To amplify the DNA, the following primers dT-primed first-strand cDNA synthesis. For measure- were used: rs437876: forward: 50-gaagacctcgcacgtcccttttg- ment of the relative level of expression of VIPR1 30reverse: 50-gtggcttgcctagtggctgtcg-30, annealing at 64 1C, in untreated and LPS-stimulated monocytes, real-time restriction enzyme: HpyCH4V (New England Biolabs, RT-PCR was performed using the 7300 Real-Time PCR Ipswich, MA, USA), rs896 and rs9677: forward: System (Applied Biosystems (ABI)). Reactions were 50-tgaagatgcagctcactaccc-30 and reverse: 50-gtgaagggcaga performed in a Micro-Amp Optical 96-well reaction cagaggag-30: annealing at 64 1C, restriction enzymes: DraI plates (ABI) using 2.5-ml cDNA of treated or untreated and EcoRI (M-Medical, Milano, Italy), respectively; monocytes, in triplicate wells, 12.5 mlof2 Master Mix rs342511: for 50-cagccaccagcgaatgctaggtccc-30 (the under- (ABI), 1.25 ml of validated Assay on Demand gene lined c has been introduced to create the restriction site) expression (ABI, HS00270351). The final volume of the and reverse 50-ggagggcagctcttgattcctg-30; annealing at PCR was 25 ml. The PCR was performed using the 60 1C; restriction enzyme: HpaII (M-Medical). Restriction following amplification scheme: one cycle of 10 min at enzyme digestions were carried at 37 1C overnight. 95 1C followed by 60 cycles of denaturation for 15 s at Fragments were separated on agarose gel or on a 10% 95 1C and an annealing/extension step of 1 min at 60 1C. polyacrylamide gel, stained with ethidium bromide and All reactions were conducted using a 7300 Sequence photographed under UV. Most likely haplotypes and LD Detector thermocycler linked to a PC using the Sequence have been estimated using the software Haploview Detector software (ABI). Glyceraldehyde-3-phosphate (http://www.broad.mit.edu/mpg/haploview/) SNP map- dehydrogenase assay on demand (ABI, HS 99999905) ping at position –13910 (SNP rs4988235) of the gene for was used to ensure quality of RNA preparation, to lactase was amplified using the following primers: account for efficiency of the reverse transcription and to forward: 50-gttagacggagacgatcacg-30; reverse: 50-gaatg check for any loading variation of the initial cDNA cagggctcaaagaac-30. Genotype was performed by minis- amount. equencing; the amplified products were treated with 0.5 U of shrimp alkaline phosphatase (SAP) (Roche Western blot analysis Applied Science, IN, USA) and 2.5 U of exonuclese I After 0, 3, 6, 9 and 12 h of culture in presence of (EXO I) (BioLabs) at 37 1C for 120 min and at 75 1C for LPS (0.05 mgml1), monocytes were washed twice with 15 min for inactivation. For minisequencing reactions, the phosphate-buffered saline and lysed in ice-cold radio- commercial fluorescent-based minisequencing kit SNaP- immuno precipitation assay buffer (50 mM Tris-HCl, pH shot multiplex (Applied Biosystems, Foster City, CA, 7.4, 1% Nonidet P40, 0.5% sodium deoxycholate, 0.1% USA) was used, with 2 pmol of primer. The sequence of SDS, 1 mM EDTA, 1 mM ethylene glycol tetraacetic acid, 0 0 the SNaPshot primer was 5 -atacagataagataatgtag-3 . 0.5 mM NaVO4,50mM NaF, 1 mM phenylmethylsulphonyl- After the extension and labelling reaction, the unincor- fluoride and a cocktail of protease inhibitors; Roche). porated dNTPs were removed by enzymatic treatment Cells debris were removed by centrifugation at 20 000 g using 0.5 U of SAP at 37 1C for 120 min and at 75 1C for for 15 min at 4 1C. Protein concentration in whole-cell 15 min for inactivation. The products were analysed on extracts was measured by Bradford assay (Bio-Rad, the ABI prism 310 Genetic Analysers. Genotyping was Munich, Germany) with bovine serum albumin used as performed by 310 ABI Prism GeneScan 2.1 software standard. Cell lysate was mixed with an equal volume of (Applied Biosystems). 2 electrophoresis sample buffer (125 mM Tris-HCl, pH 6.8, 4% w/v SDS, 20% glycerol, 100 mM dithiothreitol, Monocyte purification 0.02% bromophenol blue) and boiled for 5 min. Total Monocytes, separated from PBMC of 53 blood donors, protein extract (30 mg) for each sample was separated by were sorted using the Monocyte Isolation kit and 12% SDS–polyacrylamide gel electrophoresis and subse- Depletion Column Type LS (Miltenyi Biotec, Auburn, quently transferred to nitrocellulose membrane (Amer- CA, USA) according to the supplier’s instructions. Cells sham, Piscataway, NJ, USA). After 1 h in Tris-buffered viability as measured by Trypan blue exclusion always saline tween-20 10% non-fat milk, the membranes were exceeded 95%. Monocyte purification was higher than incubated overnight at 4 1C with a polyclonal anti-VIP 90% as assessed by flow cytometry on a FACScan flow receptor 1 antibody, a kind gift from Dr Kathleen Freson, cytometer (FACsort; Becton Dickinson, Sparks, MD, Center for Molecular and Vascular Biology, University of USA) defined by forward and side scatter properties, Leuven, Belgium41 and detected by horseradish perox- as well as by staining with anti-CD14 monoclonal idase-conjugated goat anti-rabbit secondary antibody antibody (BD Pharmingen, San Diego, CA, USA). Cells using the ECL Western Blotting Detection Kit (Amer- were seeded at concentration of 106 cells per ml and sham Biosciences). a-Tubulin monoclonal antibody suspended in RPMI 1640 supplemented with 10% (1:10.000; Santa Cruz Biotec, Santa Cruz, CA, USA) was human serum, 2 mML-glutamine, 25 U ml1 penicillin used to ensure equal protein loading. The intensity of and 25 U ml1 streptomycin (all purchased from Gibco, bands was quantified by densitometric analysis using the Invitrogen, Carlsbad, CA, USA) in cell culture plates at program UN-SCAN-IT gel (Silk Scientific Inc., Orem, UT,

Genes and Immunity Vasoactive intestinal peptide receptor 1 and HLA-B27 F Paladini et al 666 USA). Results were evaluated as the ratio of intensity of transfer in a murine model of Sjogren’s syndrome. Ann Rheum VIPR1 to that of a-tubulin from the same sample. Dis 2006; 65: 195–200. 11 Lara-Marquez M, O’Dorisio M, O’Dorisio T, Shah M, Karacay B. Selective gene expression and activation-dependent regula- Statistical analysis tion of vasoactive intestinal peptide receptor type 1 and type 2 Allele frequencies of SNP genotypes were compared by in human T cells. J Immunol 2001; 166: 2522–2530. two-tail Fisher’s exact test. Bonferroni correction for 12 Brewerton DA, Hart FD, Nicholls A, Caffrey M, James DCO, multiple comparisons was performed. The Hardy– Sturrock RD. Ankylosing spondylitis and HL-A 27. Lancet Weinberg equilibrium was tested by comparing expected 1973; 40: 904–907. to observed genotype frequencies by w2-test. Cochrane– 13 Benjamin R, Parham P. 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Population choice Acknowledgements in mapping genes for complex diseases. Nat Genet 1999; 23: 397–404. We thank the patients for their cooperation and Federica 18 Lampis R, Morelli L, Congia M, Macis MD, Mulargia A, Lucantoni for technical assistance. A special thank to Loddo M et al. The inter-regional distribution of HLA class II Dr Kathleen Freson for providing the anti-VIPR1 poly- haplotypes indicates the suitability of the Sardinian popula- clonal antibody. This work has been partially supported tion for case–control association studies in complex diseases. by Fondazione Cenci-Bolognetti, ‘Sapienza’ University of Hum Mol Genet 2000; 9: 2959–2965. Rome. 19 Fiorillo MT, Cauli A, Carcassi C, Bitti PP, Vacca A, Passiu G et al. Two distinctive HLA haplotypes harbor the B27 alleles negatively or positively associated with ankylosing spondylitis in Sardinia: implications for disease pathogenesis. 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